The objective of the proposed research is to resolve the biological[unreadable] roles and biochemical activities of two transcription factors expressed[unreadable] in the mammalian brain. Both proteins are members of the bHLH-PAS[unreadable] family, respectively designated neuronal PAS domain protein 1 (NPAS1)[unreadable] and NPAS2. NPAS1 is expressed exclusively in neurons of the brain and[unreadable] spinal cord. NPAS2 is expressed predominately in the brain and spinal[unreadable] cord but also in epithelial cell associated with the gut, uterus,[unreadable] mammary gland and prostate. A three-pronged approach will be employed[unreadable] to study the biology and biochemistry of these transcription factors.[unreadable] The first prong of attack will involve targeted disruption of the genes[unreadable] encoding NPAS1 and NPAS2 in laboratory mice. Conventional methods have[unreadable] already been used to uniformly eliminate NPAS1 function. Such mice are[unreadable] viable and an appropriately sized colony is being bred for behavioral[unreadable] and neuro-anatomical studies. NPAS2 function will be conditionally[unreadable] eliminated in each tissue known to express this transcription factor.[unreadable] Mice lacking NPAS2 in specific tissues will be investigated in[unreadable] behavioral, neuro-anatomical and physiological studies of brain tissue[unreadable] in response to both global ischemia and excitotoxic drugs. The second[unreadable] objective of the proposed research will entail efforts to identify NPAS1[unreadable] and NPAS2 target genes. Cultured neuronal cells will be programmed to[unreadable] conditionally express either NPAS1 or NPAS2 in response to defined[unreadable] stimuli. Messenger RNA will be prepared from such cells before and[unreadable] after induction of each transcription factor. This material will then[unreadable] be subjected to representational difference analysis (RDA) in order to[unreadable] search for genes that are either activated or repressed following[unreadable] induction of NPAS1 or NPAS2. The RDA method will also be utilized to[unreadable] analyze messenger RNA prepared from brain tissue derived from mutant[unreadable] mice lacking either NPAS1 or NPAS2. Once putative target genes have[unreadable] been identified, established methods of molecular analysis will be[unreadable] employed to search for NPAS1 and NPAS2 response elements. The third and[unreadable] final experimental approach will entail biochemical and biophysical[unreadable] studies focused on the mechanisms regulating activation of NPAS2. NPAS2[unreadable] exists in a latent, cytoplasmic state in pyramidal neurons of the[unreadable] hippocampus and cerebral cortex. As such, there is reason to anticipate[unreadable] that the activity of the protein as a functional transcription factor[unreadable] will require a specific inductive event. Biochemical studies of[unreadable] recombinant NPAS2 have revealed the presence of a heme prosthetic group[unreadable] associated with the PAS domain. Biochemical, molecular biological and[unreadable] biophysical studies will be employed to study the functional relevance[unreadable] of the heme prosthetic group as well as its role in controlling the[unreadable] activity of NPAS2 in pyramidal neurons.